DESCRIPTION: The investigators propose to continue their theoretical and experimental studies on the determination of segments during early Drosophila embryogenesis. They aim for a better understanding of the process by which maternal gene product gradients give rise to the stable pattern of expression of the segment polarity genes engrailed and wingless in stripes one nucleus in width. Their working assumption is that these patterns are determined by a regulatory cascade of transcription factors expressed from segmentation genes. They will employ what they call the "gene circuit method." This method takes as state variables the experimentally observable levels of transcription factors in an anterior-posterior row of cell nuclei, and uses a dynamic model of the general form dv/dt = F - G, where v is the level of transcription factor in a nucleus, F is the rate of formation of the transcription factor, and G is the net rate of loss by diffusion to and from adjacent nuclei and by decay. The model assumes that the time delay between transcription and translation has no important effect on overall kinetics, that mRNA processing and regulation have no important effect on the overall kinetics, and that effects on transcription can be treated as affecting translation (i.e. F) immediately. In general F is affected by any or all of the transcription factor levels in the nucleus (acting as transcription factors) and by the local level of maternal bicoid protein and general transcription factors. F is essentially a quasi-switch function (a nonlinear sigmoidal function) driven by a weighted sum of the local levels of the transcription factors, bicoid protein, and general transcription factors. G is a linear function of v and the concentrations of the same transcription factor in the two adjacent nuclei. During embryogenesis the nuclei divide simultaneously and periodically; and the rate of diffusion between cells increases with each division, because the distance between nuclei decreases. To fit the model to observed levels of the transcription factors, the parameters are adjusted by least-squares optimization. This optimization is carried out by the method of simulated annealing, because simpler methods tend to stop at one of the many local minima, and do not reliably give the global minimum. Simulated annealing is essentially a trial-and-error method which requires intensive computation. The aims of this project are (1) to determine the gap and pair-rule gene circuit that controls each of the stripes of expression of the 7 pair-rule genes; (2) to characterize the regulatory inputs from maternal, gap, and pair-rule genes that lead to formation of engrailed and wingless stripes, (3) to inquire whether the four-fold hierarchy of the segmentation genes is a required form of organization for forming segments in a syncytium, or an evolutionary accident, and (4) to explore the role of cell-cell signaling in regulating engrailed and wingless after gastrulation. In the first year, it is proposed to work on getting new and more extensive data and on development and testing of a parallel computation method for faster computing of simulated annealing. A number of new antisera will be prepared or obtained from other investigators. Antibodies must be raised in two or three different animals, so that each of two or three gene products can be visualized by using second antibodies to the respective Fc regions, each conjugated to a different fluorescent dye. Two methods will be developed for quantitative digital data collection: confocal microscopy and wide-field fluorescence microscopy combined with a photometric CCD array. Software will be acquired or developed for image processing and for averaging signal over each nucleus for fully quantitative measurement of expression level, and for aligning expression domains from different embryos. Expression data will be obtained for the remaining primary, secondary, and tertiary pair-rule genes, and simulated annealing will be used to analyze successively enlarged versions of the gap gene plus pair-rule gene system. It may be necessary to extend the model to allow for time delay between transcription and translation, and for changes in diffusion of some factors. Expression data will likewise be obtained and analyzed for the engrailed and wingless genes. Finally, post-gastrulation data will be obtained and analyzed with a circuit model that includes cell- cell signaling (which would allow expression in one cell to affect transcription in an adjacent cell).